Chemotherapeutic intervention has been a bulwark of modern medicine. Treatment of cancer, hypertension, heart disease, inflammation and endocrine disorders are just a few examples of human disorders, some of them life threatening, which have been successfully managed or cured by drugs alone or in combination with other therapy. Infectious diseases caused by bacteria, fungi and parasites have also been successfully managed or cured by drug therapy.
Central to the discovery of new medically effective drugs is the ability to screen for effective agents and identify sources of their production. It is safe to say that the ability to screen and locate important compounds is often the limiting step in the discovery of these agents. Current assays for drugs used in the treatment of hypertension and inflammation, for example, often require the use of unpredictable enzyme and cellular assays. Similarly, the detection of drugs that are effective against viruses often require sophisticated and cumbersome cell culture or virus enzyme assays. These assays are not often amenable to high volume studies, and most crude samples of impure compounds or crude extracts of natural products, which are often the source of effective drugs, cannot be tested in these assays without considerable prior purification.
In addition, the discovery of effective drugs depends either upon the large scale screening of thousands of different compounds or the reasoned design of drugs that, by their structure, are predicted to be effective. This approach has been, to date, the only means of drug discovery.
Much attention has focused on the discovery of therapeutic agents effective against Human Immunodeficiency Virus (HIV). HIV is the causative agent of the human disease, Acquired Immunodeficiency Syndrome (AIDS) and AIDS Related Complex (ARC). These are devastating illnesses that represent a major worldwide health threat. HIV is a member of the RNA genome-containing retroviruses and requires the function of virally specified gag and reverse transcriptase (pol) polypeptides. As with other RNA tumor viruses, the HIV life cycle involves synthesis of a double stranded DNA copy of the genomic RNA. This process is catalyzed by the viral reverse transcriptase, and inhibition of this enzyme inhibits virus replication.
Dependency of HIV upon unique virally encoded functions, such as the reverse transcriptase, presented the possibility of rationally designing antiviral agents to be directed towards interfering with those functions. For example, the nucleotide analog azidothymidine (AZT) is an effective anti-HIV agent and is presumed to work by forming chain terminating products generated by retroviral reverse transcriptase. It should therefore be possible to impede or prevent virus growth by developing pharmacological agents that inhibit essential virus functions.
The gag and pol proteins perform essential HIV retroviral functions. Mature gag and pol products are derived from the proteolytic processing of polypeptide precursors. This proteolytic cleavage is mediated by a virally specified protease that is essential for virus growth. Inhibition of this viral protease is therefore likely to prevent HIV maturation.
There is enormous interest in the discovery of HIV protease inhibitors present as natural products or derived from chemical synthesis. Each candidate inhibitor must be tested for its ability to prevent HIV protease from enzymatically cleaving substrates such as preparations of either gag-pol precursors or synthetic oligopeptides containing the HIV protease recognition sequence. Such assays are time consuming and costly, usually involving separation of cleaved products by electrophoresis or other methods of chromatography, such as HPLC. All of these methods require a reasonable degree of technical sophistication. The search for an effective HIV protease inhibitor would be greatly assisted by the availability of a simple, rapid and inexpensive assay.
Similarly, it is critical that a rapid screening assay for inhibitors of HBV be developed. HBV-is associated with chronic liver disease, including chronic hepatitis, cirrhosis and hepatocellular carcinoma. Infection with HBV can lead to one, or a combination, of the following outcomes: inapparent infection followed by seroconversion, acute hepatitis followed by recovery or death, and chronic infection. Chronically infected individuals possess HBV genetic information in their hepatocytes and often experience persistent viremia with an absence of neutralizing antibodies. These individuals may experience no frank symptoms and may carry the virus in their livers, and possibly elsewhere, for a period of years.
Twenty to forty percent of all chronic carriers will eventually die from serious liver disease, including cirrhosis and primary liver cancer. Although the precise mechanisms of these clinical endpoints are not known, these outcomes are believed to be the result of HBV infection. There is no question that HBV is associated with serious liver pathology and the use of antiviral agents in the treatment of HBV infection should be of great value.
The epidemiology of HBV indicates a worldwide problem. More than 250,000,000 people are chronic carriers of HBV. As many as 100,000,000 of these people can be expected to die prematurely from serious liver disease. Most of these people will be afflicted and require hospitalization during the most productive period of their early-mid adulthood. It is evident that a cure for HBV induced disease will relieve great suffering.
Although there is an effective vaccine to prevent HBV infection from establishing chronic infection, there is currently no effective therapy for the hundreds of millions of people infected. Recently, alpha interferon has been shown to be useful in 10-25% of adults recently infected. Although it is not helpful in those who have been infected at birth, the vast majority of carriers, the promising results of alpha interferon underscore the potential of anti-HBV therapies.
Currently, assays for the detection of anti-HBV agents are limited. Animal models of HBV, including woodchuck hepatitis (WHV) and duck hepatitis viruses (DHBV), have been of enormous value in the virological study and assay of potential chemotherapeutic agents. However, animal experiments are expensive and time consuming. Therefore, the large scale investigation of many different compounds using such methods is not practical and the assay of crude mixtures of materials is not reasonable. Further, tissue culture systems for the propagation of hepatitis viruses have been reported. Such systems are likely to prove to be of considerable value. However, even tissue cultures are delicate and those described are of very limited efficiency. Since HBV grows poorly in tissue culture and biochemical assays for the two known virally specified enzymes are complicated, the pursuit of antiviral agents has been limited. Other virus systems have benefited from the development of biochemical assays for virus-specific enzymes. At present, it is difficult to impossible to assay HBV-specific enzymes in the absence of infected cell material or partially pure virions. Clearly, HBV antiviral research would benefit from more simple, rapid and specific assays.
There is also a global need for the identification and selection of inhibitors of members of the Herpes virus family. Herpes viruses are ubiquitous and are the causative agents of a wide range of opportunistic human diseases. They are among the leading causes of non-trauma induced blindness, encephalitis, sexually transmitted diseases, and morbidity in immunocompromised hosts.
Since the isolation of antibiotics, interest in bacteria both as the source of biologically important molecules and as a solution to environmental problems has been a leading impetus for the study of these organisms. In addition to the hundreds of antibiotics, the anticancer agent, adriamycin, the antiviral agent, adenosine arabinoside, and the immune modulator, cyclosporin A have all been isolated from microorganisms. Recently, bacteria with other special properties such as the ability to dissolve oil spills or to prevent frost formation on crops have been important additions to the repertoire of useful bacteria. With the advent of recombinant DNA techniques it became clear that recombinant bacteria could also be turned into a factories for producing new and useful drugs that could not otherwise be readily synthesized in the laboratory. These drugs include such well known proteins as insulin, growth hormone and clotting factor. The use of recombinant bacteria has also been invaluable in the study of clinically important bacteria and viruses. These efforts have increased the safety of studying very hazardous organisms, and have allowed for the production high levels of proteins necessary for both biochemical studies and safe vaccines. Moreover, recombinant bacteria have the capacity to internalize a broad spectrum of chemical classes many of which are also taken up by mammalian cells.
The "AMES" test for mutagens has proven to be a useful predictor of the carcinogenic potential of many chemicals. (Ames et al., 1975, Methods For Detecting Carcinogens And Mutagens With The Salmonella/Mammalian-Microsome Mutagenicity Test, Mutat. Res., 32:347-364). This assay exploits the rapid and inexpensive growth of bacteria to test the ability of test compounds to cause mutations. The invention described herein utilizes a rapid bacteriologically based bioassay to test pharmacological compounds for their ability to inhibit activities of a plethora a gene products associated with disease states, such as proteins elaborated by viral and pathological endogenous genes.
EPO Publication No. 0 421 109 A2 (Baum et al.) also discloses a method for screening compounds which inhibit protease utilizing E. coli transformed with .beta.-galactosidase which is capable of being assayed by reacting with a color indicator compound to produce a color change. In this method, a DNA sequence encoding a peptide sequence recognized by HIV or polio 3C protease was inserted into the .beta.-galactosidase gene, and this chimeric construct was transformed into an E. coli strain, along with an HIV or polio 3C protease gene. Growth of recombinant organisms was carried out in a nutrient medium containing the chromogenic compound and a potential protease inhibitor compound. If the test compound inhibits the protease, the expression system produces an intact chimeric .beta.-galactosidase, and color is maintained in the assay system. If the compound is not an inhibitor, the protease cleaves the protease enzyme cleavage site of the peptide, the reporter protein is inactivated and no color is observed in the assay system.
The instant invention provides a method in which dominant selectable markers are used for the selection of mutant microorganisms which may themselves be producing valuable compounds or possess valuable mutations. Unlike Baum et al., the present invention may utilize a chromogenic or non-chromogenic indicator compound and thus has greater utility. Further, nowhere in Baum et al. is it disclosed that the tetracycline resistance protein, which is useful in the instant invention, can be used as the reporter protein. Additionally, the method of Baum et al. is limited to the insertion of an HIV or 3C protease cleavage site sequence into the .beta.-galactosidase gene. There remains a need for rapid, simple and inexpensive methods to identify and isolate viral inhibitors and other medically important agents.